This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The preprophase band (PPB) is an array of microtubules (MTs) that marks the site on the plant cell plasma membrane where the cell plate will fuse during the final stage of cytokinesis. We have investigated the mechanism of PPB membrane modifications in epidermal cells of cryofixed onion cotyledons by analyzing the endocytic and secretory structures associated with PPB formation in serial thin sections and electron tomograms. The endocytic structures that were quantitatively evaluated in this study included clathrin-coated pits and vesicles, as well as structurally related non-coated vesicles. All of these structures were located within 0.4 [unreadable]m of the plasma membrane and contained characteristic darkly staining cargo molecules. A causal relationship between these structures was demonstrated by short-term treatment with brefeldin A (BFA), a secretory and endocytic pathway inhibitor. Under the conditions used, only a fraction of the Golgi stacks showed BFA-induced structural changes. In contrast, the treatment reduced dark-core endocytic pits by >90%, the number of cortical, clathrin-coated, dark-core vesicles by ~80%, and the number of cortical, non-coated, dark-core vesicles by ~67%. During PPB formation, the number of dark-core, endocytic vesicles adjacent to the forming PPB was 2.6-fold higher than in the cytoplasm underlying the non-PPB regions of the plasma membrane. In contrast, the secretory activities, as monitored via the number of horseshoe-shaped profiles in the plasma membrane, were similar between the two regions. Based on these findings, we propose that the modification of the plasma membrane at the site of the PPB involves removal of selected membrane molecules by clathrin-coated pits and vesicles. Karahara, I., J. Suda, T. Murata, L.A. Staehelin and Y. Mineyuki (2006) Preprophase band formation involves endocytosis mediated by chlathrin-coated pits and vesicles. Submitted to Plant Cell